Mouse Tumor-Associated Endothelial Cells-CT26

Ultra-High Concentration Nitric Oxide Enhances PD-L1 Expression and Synergizes with Immune Checkpoint Blockade

Immune checkpoint inhibitors (ICIs) achieve durable responses in only a subset of patients. Building on prior work showing that ultra-high-concentration gaseous nitric oxide (UNO) stimulates antitumor immunity when combined with surgery, Confino et al. investigated whether UNO enhances the efficacy of PD-1 blockade.

CT26 colorectal cancer cells were treated with 25,000-100,000 ppm UNO in vitro for 10-60 seconds. Annexin V/PI staining showed time- and dose-dependent apoptosis: 30 seconds of 100,000 ppm UNO caused near-complete cell death (99.6%), and 1 minute of exposure at all levels resulted in >79% apoptosis (Fig. 1A). Specificity was confirmed in that exposure to nitrogen gas had no effect (Fig. 1B). UNO treatment also upregulated PD-L1 expression on viable and early apoptotic cells. A 10-second exposure to 100,000 ppm UNO increased PD-L1 positivity from 70.9% to 85.1%; extending exposure to 30 seconds raised this to 96.7%, with lower concentrations also inducing significant upregulation (Fig. 2A). One-minute exposures at all doses maintained PD-L1 expression above 94%. Nitrogen exposure did not increase PD-L1 and instead reduced baseline levels (Fig. 2B). Similar effects were observed in 4T1 triple-negative breast cancer cells, albeit requiring higher UNO thresholds. These results reveal that short-term exposure to UNO preferentially kills tumor cells and up-regulates PD-L1 in surviving cells. That double whammy could turn immunologically "cold" cancers "hot," making them more vulnerable to PD-1 inhibitors. These findings, together with previously reported UNO-mediated increases in tumor-infiltrating T cells, dendritic cells, and B cells, suggest UNO as a potent adjuvant to boost ICI efficacy.